manufacturer :       BioRad

model :                   Eluter

annotation :           Dokumente engl.

The Whole Gel Eluter provides an alternative method for recovering samples from SDS and Native PAGE slab gels. It is a preparative electrophoresis instrument which allows simul­taneous electro-elution of multiple protein bands separated on a polyacrylamide gel. Elution is in the transverse direction, through the thickness of the gel. Proteins are eluted into narrow chambers with each of these fractions containing individual or multiple protein bands.

The eluter consists of a solid acrylic base, a drop-in bottom electrode, an elution cham­ber core, an upper plate/spring electrode and a lid. The Whole Gel Eluter comes in two sizes. The large format accommodates slab gels up to 20 cm long and the small format is designed for mini-gels (Mini-PROTEAN® II and Ready Gels). Filter paper and a cellophane sheet are placed between the bottom electrode and the elution chamber core. The gel is sandwiched between the top of the elution chamber core and the spring electrode. During elution, the pro­teins are captured in one of the 30 elution chambers of the large eluter or one of the 14 cham­bers of the mini eluter.

One important application for this instrument is the purification and subsequent direct screening of protein mixtures. Andersen and Heron describe a direct cellular analysis of a complex protein mixture for biological activity.1 The eluter acts as an electrodialyzer remov­ing SDS from protein, leaving them in a non-toxic physiological buffer that can then be used directly in a cellular assay. The Rotofor® and Prep cell are recommended for larger scale elec­trophoretic protein purification.

Simple procedures are provided in this manual for optimizing elution conditions. It is recommended that these procedures be performed for each new sample and when making changes to slab gel electrophoresis conditions.

What Is a Whole Gel Eluter?

The whole gel eluter and mini whole gel eluter are unique electroelution tools that simultaneously elute and collect multiple bands from whole polyacrylamide gels. These fast, efficient systems eliminate the tedious, time-consuming steps of slicing and eluting gel sections. They simply elute all biomolecules from the gel into liquid fractions.

Harvesting Box

Vacuum adjustment valve allows controlled collection of fractions

Simultaneously collects up to thirty 3 ml fractions or fourteen 0.5 ml fractions in seconds

Rack is numbered for easy identification of the fractions

Upper Plate Assembly

Spring-loaded electrode accommodates gels up to 3.0 mm thick

Preparative Gel

Allows preparative quantities of biomolecules to be eluted

Elution Chamber Core

Closely spaced, precision machined channels provide maximum coverage of the gel surface

Ports for manual or vacuum-assisted harvesting

Base Assembly*

Durable platinum-coated titanium plate electrodes

Leveling feet easily adjust for system adaption to uneven surfaces

*Filter papers and cellophane not shown

Rapid Recovery of Separated Proteins, DNA or RNA

Finding your biomolecule in a complex cell lysate can be a slow, challenging procedure. And recovering it in an active state can seem next to impossible. That's why whole gel elution is such a welcome addition to the big picture of preparative electrophoresis.

Whole gel eluting rapidly extracts all biomolecules from an entire preparative gel (SDS- or native PAGE). Individual bands or groups of closely spaced bands are collected in liquid fractions, quickly and easily making samples ready for subsequent analysis or bioassay.

Choose the Whole Gel Eluter That's Right for Your Application

Whole gel eluters accommodate a variety of gel sizes. The whole gel eluter is ideal for standard size gels, 14 x 16 cm or larger. This provides the capacity necessary for loading milligram quantities of proteins. The mini whole gel eluter is perfect for rapidly studying smaller amounts of biomolecules in large or mini formats. Both handcast and precast mini gels fit the mini whole gel eluter. Fractions can be collected manually or with the optional vacuum assisted harvester.

Five Simple Steps to Preparative Elution:

Resolve proteins by vertical PAGE

Cut gel to size

Place gel on whole gel eluter

Apply electrical field

Collect fractions

Screening Biomolecules

The whole gel eluter can screen complex mixtures of proteins, DNA, or RNA in cell assays. Eluted fractions are in nontoxic form, immediately available for in vivo or in vitro testing. As demonstrated in the figures below, the whole gel eluter has been used for direct mapping of antigenic fractions from Mycobacterium tuberculosis. Fractions that held T-cell antigens were identified, and single proteins from short-term culture filtrates (ST-CF) were then purified. For more information, request bulletin 2043.

M. tuberculosis culture filtrate was divided into narrow, sharply defined fractions on the whole gel euter. Culture filtrate (5 mg) was separated in a 10–20% SDS-PAGE gel, and the gel was electroeluted at 40 V for 20 min on the whole gel eluter. The fractions were analyzed on a 10–20% SDS-PAGE gel, and proteins were visualized by silver staining. Lane F, ST-CF; lanes 1–26, fractions 1–26 off the eluter. No proteins were detected in lanes 27–30.

Stimulation of T cells in vitro with the M. tuberculosis fractions. IFN release from lymphocytes stimulated in vitro with a panel of ST-CF fractions. The lymphocytes were isolated from the spleens of TB-infected mice and stimulated for 48 hr with the fractions. Cytokines in the culture supernatant were measured by ELISA. All values are given as mean of triplicates ±SEM, and were obtained with cells pooled from 3–5 mice.

Purifying Biomolecules

When the biomolecule of interest is well resolved on a PAGE gel, it can be isolated by the whole gel eluter and made available in liquid form for antibody production, sequencing, or characterization studies.

Dave Armbruster from Ohio State University, Dept. of Microbiology, has isolated mature RNA (approximately 182 nt) from preRNA and other total cellular components with the whole gel eluter. He loaded approximately 1 mg of total cellular RNA on an 8 M urea preparative 6% acrylamide gel, and then eluted the resolved bands in 1x TBE buffer. After analyzing 30 fractions on a northern blot, three fractions contained enough pure, mature RNA for RT-PCR, cloning, and sequencing.

The distance between biomolecules resolved on a PAGE gel must be greater than 5 mm for the mini whole gel eluter, and 6 mm for the whole gel eluter to ensure the biomolecule of interest is eluted in its pure form without contamination from neighboring bands.

For quick determinations, both eluters include a ruler indicating channel distances where specific bands will elute.

One Step in a Preparative Puzzle

Whole gel eluters are additional components to the expanding line of preparative electrophoresis products from Bio-Rad. They complement the Rotofor preparative isoelectric focusing devices, and the prep cell preparative electrophoresis instruments. With the Rotofor cells, extraneous proteins in complex samples can be removed prior to screening with the whole gel eluter. Once an active biomolecule has been identified with an eluter, it can be purified in larger quantities with a prep cell.

To put it simply, the whole gel eluter greatly increases the power and capabilities of vertical electrophoresis.

The whole gel eluter system is available in 14-well and 30-well versions with optional collectors and includes consumables for up to 25 runs.

Specifications Mini Whole Gel Eluter